Method for the improvement of neuronal regeneration

ABSTRACT

A method for the improvement of neuronal regeneration by prevention or inhibition of basal membrane formation induced by a lesion of neuronal tissue.

The present invention refers to a method for the improvement of neuronalregeneration, a medicament for the improvement of neuronal regenerationand use of a specific inhibitor substance.

Injury to adult mammalian CNS fiber tracts leads to the formation of alesion scar consisting of a convoluted fringe of astroglial processeslined by a basal membrane (BM). This lesion scar is implicated as amajor extrinsic constraint to effective axon regeneration in brain andspinal cord (1-4). While the dense astrocytic network is a permissivesubstrate for axon growth (5, 6), the presence of BM has beenhypothesized as a crucial impediment for regeneration (7). However,experimental evidence was not shown. To the contrary, when the BM formedafter a lesion of neuronal tissue was removed (24), no improvedregeneration could be reproducibly monitored (25). Therefore, it isstill of great importance to have a method for improving regeneration ofinjured neurons.

WO 93/19783 discloses a method for preventing, supressing or treating aCNS pathology characterized by a deleterious accumulation ofextracellular matrix in a tissue by contacting the tissue with an agentthat inhibits the extracellular matrix producing activity of TGF-β. Thedisclosed methods can be used to prevent, suppress or treat scarformation in the CNS. As useful agents are addressed neutralizinganti-TGF-β antibodies, Arg-Gly-Asp-containing peptides, decorin and itsfunctional equivalence such as biglycan and TGF-β antagonists. TGF-β hasa wide spectrum of physiological functions such as activation of cell ofthe immune system, inhibition of cell proliferation, neurotrophiceffects on sensory neurons, inhibition of Schwann cell myelination,anti-profilerative effects on glial cells, immunsuppressive effects,stimulation of extracellular matrix deposition and chemoattraction ofmicroglia cells. The anti-TGF-β treatment would induce the oppositeeffects. Inhibition of TGF-β activity leads to numerous non-specificcellular responses, which may even lead to unwanted side effects. Oneobject of the invention is to avoid such potential unwanted sideeffects.

Surprisingly, improvement of regeneration of neuronal tissue afterlesion is achieved by a method of the present invention.

According to the method of the invention improved regeneration ofinjured neuronal tissue is achieved by specific prevention or specificinhibition of basal membrane formation induced by a lesion of neuronaltissue.

The basal membrane is a structure which is composed of differentelements. Elements of the basal membrane are collagen IV, laminin,entactin (Nidogen) accessory substances. The assembly of the elements tothe basal membrane is performed by enzymes which may be assisted bycofactors.

Inhibitors of TGF-β are not involved with a specific prevention orspecific inhibition of basal membrane formation induced by lesion ofneuronal tissue. According to the present invention it is achieved in anadvantageous manner that a specific interaction is provided.

Preferably, the formation of the basal membrane is prevented orinhibited by applying a specific inhibitor substance of the synthesis ofbasal membrane building elements, or the assembly of basal membranebuilding elements, or both the synthesis of basal membrane buildingelements and the assembly of basal membrane building elements to a bodyin need thereof. The building elements of the basal membrane are inparticular those which are involved with the formation of the basalmembrane, for instance molecular structures building up the basalmembrane, such as monomeric compounds, accessory substances, substancesfor the assembly of the components of the basal membrane and the like.

In particular, the basal membrane building elements are selected fromthe group consisting of collagen IV, laminin, entactin, accessorysubstances for proper function, or the assembly of the basal membrane,or both the proper function and the assembly of the basal membrane.

A specific inhibitor substance of the invention is capable of preventingor inhibiting the formation of the basal membrane and/or is specificallyinterfering with the assembly process of the basal membrane. Preferably,the specific inhibitor substance is selected from the group consistingof antibodies against collagen IV, laminin, entactin, accessorysubstances for proper function, or the assembly of the basal membrane;Fe-chelating agents; inhibitors of amino acids hydroxylases, such asprolyl-4-hydroxylase, lysine-hydroxylase; 2-oxoglutarate competitors;antisense nucleotides or nucleotide analogs which are able to prevent orinhibit the expression of basal membrane building elements, and thelike.

According to the invention can further be used those inhibitorsubstances which are selected from the group consisting ofN-oxaloglycine; Zn salts; pyridine derivatives, such as5-arylcarbonyamino- or 5-arylcarbamoyl-derivatives, 2-carboxylate, 2,5dicarboxylate, their ethyl esters or ethyl amides or -5-acylsulfonamides, 2,4 dicarboxylate, their ethyl esters or ethylamides, ordimethoxyethylamides; 3,4 bipyridine, such as 5 amino-6-(1H)-one,1,6-dihydro-2-methyl-6-oxo-5-carbonitril; 2,2′-bipyridine, such as5,5′-dicarboxylic acid or its pharmaceutically acceptable salts,4,4′-dicarboxylic acid ethyl ester or ethyl amide;3,4′-dihydroxybenzoate, such as the diethyl ester; proline and itsstructural and functional analoges; β-aminopropionitrile;desferrioxamine; anthracyclines; 2,7,8-trihydroxy anthraquinones,fibrostatin-C; coumalic acid or its pharmaceutically acceptable salts;5-oxaproline, β-lactam antibiotics.

In a preferred embodiment of the present invention the specificinhibitor substance(s) are applied in combination with one or moresubstances being capable of stimulating neuronal growth or inducing theexpression of growth promoting proteins. Such neuronal growthstimulating substances are neurotrophic growth factors of theneurotrophin family and other growth factor families such as fibroblastgrowth factors, insulin and insulin-like growth factors, as well asepidermal growth factor, ciliary neuronotrophic growth factor (CNTF),glial cell-derived growth factor (GDNF), cytokines, neurotrophicproteoglycans and gly-cosamino-glycans, neural cell adhesion moleculeslike L1 (NILE), growth-associated proteins like GAP43 and anti-apoptoticproteins like bcl-2.

According to the invention it is preferred to locally apply the specificinhibitor substances in the neuronal tissue, intraventricularly, orsystemically, in particular orally or intravenously.

The concentration of the specific inhibitor substance varies in view ofthe chemical nature. For example, antisense inhibitor substances mayhave more specific effects so that lesser amounts can be applied.

Typically, the specific inhibitor substance is applied intherapeutically effective amounts, such as 1 ng/kg to 1 mg/kg bodyweight, when low molecular compounds such as bipyridyl-derivatives areapplied.

The invention also provides a medicament for the improvement of neuronalregeneration comprising a therapeutically effective amount of a specificinhibitor substance which is capable of prevention or inhibition ofbasal membrane formation induced by a lesion of neuronal tissue.Appropriate specific inhibitor substances are described hereinabove. Themedicament may further comprise carrier substances or adjuvants in orderto facilitate an appropriate application. The medicament may furthercomprise substances which are capable of stimulating neuronal growth.

The specific inhibitor substances which are capable of prevention orinhibition of basal membrane formation induced by a lesion of neuronaltissue can be used for the manufacturing of a medicament of theinvention.

FIG. 1: Expression of collagen IV and axonal sprouting after transectionof the postcommissural fornix in untreated animals (b-d) and afterinjection of anti-Coll IV (e) or DPY (f) at two weeks postsurgery. a,Sagittal view of the adult rat brain showing the course of the fornixand the location of the transection site. Marked deposition of collagenIV in the lesion site (arrow) and proximal stump (P) of an untreatedanimal at low (b) and high magnification (c). Note, the fine structureand the spatial orientation of collagen IV deposits perpendicular to thetrajectory of the tract. d, In untreated animals regrowing fornix axonsstop sharply at the lesion site (arrow). Collagen IV deposition ismarkedly reduced in the lesion site after anti-Coll IV (e) or DPYinjection (f). Scale bars, 100 μm.

FIG. 2: Regeneration of transected fornix fibers across the lesion sitein rats treated with anti-Coll IV (a, c, e) or DPY (b, d, f) at 6 weekspostsurgery. Sagittal serial sections reacted forNF-immunohistochemistry show that in both experimental groups fiberstraverse the former lesion site (arrows) (c, d) and elongate within thedistal stump (e, f) up to the mammillary body (MB). Scale bars, 100 μm.

FIG. 3: Recovery of structural features of the regenerating fornixtract. a, b Anterograde tracing with biocytin of an anti-Coll IV treatedanimal at 6 weeks postsurgery reveals the large number of regeneratingaxons (a), their elongation within the former pathway (a) and their finevaricose morphology (b). c, Large WGA-HRP-filled axon (arrowhead) in themammillary body surrounded by compact myelin (arrows). d, e Electronmicrographs of anterogradely WGA-HRP-labeled presynaptic terminals(arrow-heads) in the mammillary body at 6 weeks after anti-Coll IVtreatment. Scale bars, 100 μm (a), 50 μm (b), 0.1 μm (c), 0.5 μm (d), 1μm (e).

FIG. 4: Electrophysiological properties of fornix fibers in unlesionedrats and lesioned/injected animals with regeneration. a, Schematicillustration showing the location of the stimulating (S) and recording(R) electrode at various conditions. b, Characteristic recordings ofextracellular action potentials in a sagittal slice prepared from ananimal with regeneration. Recordings were obtained under conditions asillustrated in a. Application of Tetrodotoxin (TTX) blocks thestimulus-evoked response. The net action potential is shown in trace 5.c and d, Distribution of conduction velocity and action potentialresponse amplitude in unlesioned and lesioned/injected animals withregeneration.

The mechanically transected postcommissural fornix of the adult rat, aunidirectional and well-characterized fiber tract (8, 9), was used todetermine whether specific biochemical or immunochemical modulation ofBM formation would provide a means to stimulate axon regeneration. Herewe report that lesion-induced BM deposition can be significantly reducedby local injection of anti-collagen IV antibodies or an dipyridyl, aninhibitor of collagen triple helix formation and synthesis. Reducing thecollagen network allowed massive axon elongation across the lesion site.The regenerating fornix fibers followed the original pathway,reinnervated their appropriate target, the mammillary body, wereremyelinated and attained nearly normal conduction properties. onfailure of adult mammalian CNS axons we examined its spatio-temporaldistribution pattern after penetrant CNS lesion and determined whetherits remodelling allows structural and functional regeneration of atransected CNS fiber tract.

The left postcommissural fornix was stereotactically transected in adultWistar rats FIG. 1 a) and the postlesion deposition of BM was analyzedusing antibodies against collagen IV (Coll IV) and laminin (LN), themajor and unique components of BM (10, 11). By the end of the secondweek after lesion the center of the wound was filled by Coll IV- andLN-rich BM (FIGS. 1 b, c). These newly formed BM were either arranged inlong continuous layers or associated with numerous blood vessels. Withinthe center of the wound the BM layers formed a parallel array alignedperpendicular to the course of the fiber tract (FIGS. 1 b, c). In thevicinity of the transected stumps, however, BM layers were deposited ashook-like turns extending along the longitudinal tract axis for about200 μm into the fornix stumps.(FIG. 1 c). In parallel with thedeposition of the BM, sprouting axons in the proximal stump reached thelesion site. They failed to cross or bypass it but stopped growing atthe wound border at about 2 weeks after lesion (FIG. 1 d). Thespatio-temporal coincidence of BM formation with the abrupt axonalgrowth arrest at the tract-lesion border strongly suggests that thenewly formed perpendicular layers of BM could be a physical impedimentfor regenerating axons.

In an effort to modulate postlesion BM deposition, either polyclonalantibodies against collagen IV (anti-Coll IV; n=14) or the iron chelatora, a′-dipyridyl (DPY; n=9) were injected locally into the lesion centerimmediately after transection. DPY is a competitive inhibitor of prolyl4-hydroxylase (12) and has been shown to prevent collagen triple helixformation (12), which results in feedback inhibition of procollagensynthesis. (13) and enhanced procollagen degradation (14). Controlanimals received a PBS injection (n=9) or were sham operated (n=3).Basal membrane formation was studied in response to antibody and drugtreatment using immunohistochemical methods. Animals receiving a singleinjection of anti-Coll IV (80-160 ng) or DPY (1.6-16 μmol) showed amassive and specific reduction in Coll-immunopositive laminae and bloodvessels in the lesion center and the fornix stumps at all examinedsurvival time points. At 2 weeks after lesion+injection only a verysmall number of Coll-immunreactive structures perpendicular to the tractcourse had developed (FIGS. 1 e, f). Control animals, however, exhibiteddense BM deposition as previously described for lesion only animals. Theapplied substances reduced the deposition of BM at the lesion site butdid not affect the number or the distribution of vascular BM in thesurrounding neuropil. Therefore, we conclude that the lesion-induced BMformation can be specifically reduced by immediate application of eitheranti-Coll IV antibodies or DPY.

To determine whether reduction of BM deposition would permitregeneration of transected axons across the lesion site, we studied theelongation of fornix axons after anti-Coll IV or DPY treatment usingimmunocytochemical staining. While sprouting fornix fibers in controlanimals ceased growing at the proximal stump-lesion interface (FIG. 1 d)large numbers of axons entered and traversed the lesion center between 2and 4 weeks after lesion+injection in those animals receiving anti-CollIV (n=11) (FIGS. 2 a, c, e) or DPY treatment (n=6) (FIGS. 2 b, d, f).Most regenerating axons formed a loop over the lesion site, entered thedistal stump and continued in a parallel bundle of fine and beaded axonswithin their previous pathway (FIGS. 3 a, b). They reached theirappropriate target, the mammillary body, at about 4-6 weeks postsurgery.Anterograde tracing with WGA-HRP into the subiculum, the origin of thefornix (not shown), or biocytin application into the proximal fornixstump (FIG. 3 a) provided proof, that the vast majority of fibers emergefrom the formerly transected fornix tract. All regenerating fornix axonsremained within their original pathway and did not invade thesurrounding neuropil. The present results demonstrate that the failureof postcommissural fornix regeneration in rat brain, in fact, dependsupon the formation of an axon growth-inhibiting BM at the lesion sitethat is oriented perpendicular to the tract course. Reduction of BMdeposition seems to be a prerequisite but also a sufficient conditionfor the transected axons to regenerate across the lesion site.

Further preferred embodiments for restitution of functional circuitryafter traumatic CNS lesion are the remyelination of regenerated fibers,the re-establishment of synaptic connections with the appropriate targetand the restoration of normal conduction properties. Structural andfunctional properties of the regenerating axons were investigated usingimmunohistochemical, morphological and electrophysiological methods.Immunohistochemistry with an antibody against myelin basic proteindemonstrated the remyelination of regenerated fornix axons along theirentire length as early as 4 weeks after surgery (data not shown). Thisobservation was confirmed by ultrastructural analysis of anterogradelyWGA-HRP labeled axons in the distal stump which showed clear evidence ofcompact myelin sheath formation (FIG. 3 c). In addition, ultrastructuralstudies provided evidence for the re-establishment of synapticconnections of regenerating axons within the mammillary body. Tracerreaction product was identified in presynaptic profiles with roundvesicles that formed asymmetric synaptic junctions at unlabeleddendrites (FIG. 3 d, e). The ultrastructural features of the labelledpresynaptic profiles correspond to those described for the RA-type(round, asymmetric) of synaptic terminal, which is considered to be ofsubicular origin (8). The electro-physiological properties of regenerated fibers were, studied using extracellular in vitro recording techniquesapplied to sagittal brain slices (400 μm) of 8 unlesioned rats and 4treated animals showing regenerated fiber tracts. In unlesioned animalselectrical stimulation of the fornix fibers elicited an extracellularaction potential with an amplitude of 1.02±0.14 mV and a conductionvelocity of 0.48±0.05 m/s (mean±SEM, n=16, FIG. 4 b-d) This axonalconduction velocity corresponds well to previously reported measurements(about 0.5 m/s for hippocampal Schaffer collaterals (15). Similar valuesfor action potential amplitude and conduction velocity (1.12±0.21 mV,0.46±0.1 m/s, n=5) were obtained in regenerating animals when thestimulating (S) and the recording (R) electrodes were positionedproximally to the lesion site (see S1 and R1 in FIG. 4 a). In the latteranimals, functionally intact fibers showing normal extracellular actionpotential amplitude and conduction velocity could also be demonstratedacross (S3 and R3 in FIG. 4 a; 0.8±0.29 mV, 0.54±0.14 m/s, n=3) anddistal to the lesion site (S2 and R2 in FIG. 4 a; 0.91±0.24 mV,0.43±0.06 m/s, n=4) (FIGS. 4 c, d). In all animals, the stimulus-evokedextracellular responses were blocked by Tetrodotoxin, confirming theirnature as Na+-dependent action potentials (FIG. 4 b). From these data weconclude that the reorganization of the fornix tract is accompanied bystructural and functional recovery of the regenerated axons.

Our results demonstrate that structural and functional restoration oflesioned mature fornix pathway can be achieved by reduction of BMformation in the lesion site. Data described here underscore theimportance of extrinsic determinants in axonal regeneration but alsodemonstrates that once the axons have crossed the lesion scar otherpotential extrinsic regeneration constraints, like CNS myelin andoligodendrocytes (9, 16-18), dense astrogliosis (6) and sulfatedproteoglycans (19, 20), do not impede their progress. The resultsfurther indicate that similar to other CNS circuits (21, 22), fornixaxons have an innate potential for regeneration and self-organization.These results give rise to new and promising concepts for therapeuticstrategies that might contribute to the reduction of neurologicaldeficits after CNS lesions.

The following examples are intended for further illustration of theinvention but are not limiting.

Surgery. The left postcommissural fornix of 42 Wistar rats (180-210 g)was transected stereotactically at a distance of about 1 mm proximal tothe target, the mammillary body, using a Scouten wire knife as,described previously (9). The completeness of transection was confirmedby serial reconstruction of the lesion site for each of the animals.Immediately after transection animals received a topical application(1.6 μl) of either polyclonal antibodies against collagen IV (anti-CollIV, Biogenex, 50-100 μg/ml, n=14) or the iron chelator a, a′-dipyridyl(DPY, 1-10 mM, n=9). Substances were pressure injected (injection time10 min) directly into the lesion site via a micropipette coupled to amicrosyringe. Controls received equal amounts of phosphate-bufferedsaline (n=9) or sham operation (n=3).

Anterograde tracing was performed for analysis of fiber course,ultrastructural morphology and target reinnervation. After a survivaltime of 6 weeks, anti-Coll IV-treated animals (n=4) received twoinjections of a 2% (w/v) solution of wheat-germ-agglutinin-HRP (WGA-HRP)into the left subicular complex (dorsal and caudal pole). Rats wereperfused 3 days later with 2% paraformaldehyde and 2% glutaraldehyde in0.1M phosphate buffer. Vibratome sections were reacted for WGA-HRP usingtetramethyl-benzidine as substrate (23).

Electron microscopy. For ultrastructural analysis vibratome sections ofanti-Coll IV-treated animals were reacted for WGA-HRP, immersed for 12 hin 1% osmium tetroxide and embedded in epon. Ultrathin sections wereexamined using a Hitachi H600 electron microscope.

Immunohistochemical staining. After a survival time of 4 days (d), 6 d,2 weeks (w), 4 w and 6 w after surgery brains were removed, frozen inisopentan (−50/−60° C.) and cut into serial sagittal 10 μm thicksections. Sections were fixed with acetone (−20° C.), preincubated in 3%H2O2 (v/v) in methanol to block endogeneous peroxidase, followed by PBScontaining 3% (v/v) normal horse or normal goat serum to reduceunspecific staining and then incubated with one of the following primaryantibodies: polyclonal anti-collagen IV (anti-Coll IV, Biogenex, 1:3),polyclonal anti-laminin (anti-LN, Biogenex, 1:5) or monoclonal cocktailagainst phosphorylated neurofilaments (anti-NF, Affinity, 1:800).Following, avidin-biotin-peroxidase complex staining (Vector Labs) wasdone using standard procedures. For evaluation of remyelination brainswere fixed with 4% paraformaldehyde, paraffinized, cut into 3-μm thickserial sagittal sections, deparaffinized and incubated as describedabove with a polyclonal anti-myelin basic protein (anti-MBP, Biogenex,1:2) or anti-NF as primary antibodies. Specificity of the stainings wasconfirmed by omission of the primary antibody.

Electrophysiology and biocytin injections. Sagittal slices of 400 μmthickness were cut on a vibratome and maintained at 34-35° C. in aninterface-type recording chamber. Artificial cerebrospinal fluid (ACSF)consisted of (in mM) 124 NaCl, 3 KCl, 1.25 NaH2PO4, 1.8 MgSO4, 1.6CaCl2, 26 NaHCO3 and 10 glucose with a pH of 7.4 when saturated with 95%O2-5% CO2. Stimuli: 100 μs, 5-20 V were delivered via a bipolar tungstenelectrode. Extracellular action potentials were registered with arecording electrode (3-5 MW) located in the middle of thepostcommissural fornix. Tetrodotoxin (TTX, Sigma) was applied locally ina concentration of 10 μM (dissolved in ACSF) with a broken micro-pipetteplaced on the slice surface near the recording site. Injections of asmall biocytin (Sigma) crystal into the fornix were performed with aminiature needle. After an incubation period of 8-10 h in the interfacechamber, slices were fixed in 4% paraformaldehyde, resectioned andreacted with ABC peroxidase reagent (Vector Labs).

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1-17. (canceled)
 18. A method of enhancing regeneration of injuriescaused by a lesion of neuronal tissue comprising local administration ofan inhibitor of basal membrane formation to enhance axonal regenerationby specific inhibition of basal membrane formation induced.
 19. Themethod according to claim 18, wherein the inhibitor is selected from thegroup consisting of antibodies against collagen IV; Fe-chelating agents;inhibitors of amino acids hydroxylases, such as prolyl-4-hydroxylase,lysine-hydroxylase; 2-oxoglutarate competitiors; antisense oligonucleotides or oligo nucleotide analogs; siRNAs; aptamers.
 20. Themethod of claim 19, wherein the inhibitor is selected from the groupconsisting of pyridine derivatives, such as 5-arylcarbonylamino- or5-arylcarbamoyl-derivatives, 2-carboxylate, 2,5, dicarboxylate, theirethyl esters or ethyl amides, or dimethoxylamides; 3,4′-bipyridine, suchas 5 amino-6-(1H)-one, 1,6-dihydro-2-methyl-6-oxo-5-carbonitril;2,2′-dipyridine, such as 5,5′-dicarboxylic acid or its pharmaceuticallyacceptable salts, 4,4′-dicarboxylic acid ethyl ester or ethyl amide;3,4′-dihydroxybenzoate, such as the diethyl ester; proline and itsstructural and functional analoges; β-aminopropionitrile;desferrioxamine; desferasirox; anthracyclines; 2,7,8-trihydroxyanthraquinones, fibrostatin-C; coumalic acid or its pharmaceuticallyacceptable salts; 5-oxaproline, β-lactam antibiotics.
 21. The method ofclaim 18, wherein the inhibitor is administered in combination with asubstance that stimulates neuronal growth.
 22. The method of claim 19,wherein the inhibitor is administered in combination with a substancethat stimulates neuronal growth.
 23. The method of claim 20, wherein theinhibitor is administered in combination with a substance thatstimulates neuronal growth.
 24. The method of claim 18, wherein theinhibitor is administered in an amount in an amount of 1 ng/kg to 1mg/kg body weight.
 25. The method of claim 19, wherein the inhibitor isadministered in an amount of 1 ng/kg to 1 mg/kg body weight.
 26. Themethod of claim 20, wherein the inhibitor is administered in an amountof 1 ng/kg to 1 mg/kg body weight.
 27. The method of claim 21, whereinthe inhibitor is administered in an amount of 1 ng/kg to 1 mg/kg bodyweight.
 28. The method of claim 22, wherein the inhibitor isadministered in an amount of 1 ng/kg to 1 mg/kg body weight.
 29. Themethod of claim 23, wherein the inhibitor is administered to an amountof 1 ng/kg to 1 mg/kg body weight.